QC Report

	general
Report generated at	2025-02-25 03:26:50
Title	AdrenalGland
Description	ATAC-seq data from Human ENCODE adrenal gland data processed in 2025
Pipeline version	v2.2.0
Pipeline type	atac
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	537908128	145594466
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	527417446	139999369
Mapped Reads (QC-failed)	0	0
% Mapped Reads	98.0	96.2
Paired Reads	537908128	145594466
Paired Reads (QC-failed)	0	0
Read1	268954064	72797233
Read1 (QC-failed)	0	0
Read2	268954064	72797233
Read2 (QC-failed)	0	0
Properly Paired Reads	516592006	137227690
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	96.0	94.3
With itself	521534888	138536038
With itself (QC-failed)	0	0
Singletons	5882558	1463331
Singletons (QC-failed)	0	0
% Singleton	1.0999999999999999	1.0
Diff. Chroms	518121	124955
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	232088452	61152820
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	48575280	9574816
Paired Optical Duplicate Reads	1070803	589818
% Duplicate Reads	20.9296	15.6572

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	483685414	130786411
Rm = Number of Mitochondrial Reads	47433360	9841071
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.0893083850957978	0.06997971420692863

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	364041376	101578580
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	364041376	101578580
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	364041376	101578580
Paired Reads (QC-failed)	0	0
Read1	182020688	50789290
Read1 (QC-failed)	0	0
Read2	182020688	50789290
Read2 (QC-failed)	0	0
Properly Paired Reads	364041376	101578580
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	364041376	101578580
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.4406882560202192	0.4105701927246308
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	1.985576029673017	1.7470083255726019
NFR / mono-nuc reads (QC pass)	False	False
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]	out of range [2.5, inf]
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	True	True

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.6001049616953431	0.5810448620171693
Fraction of Reads in blacklist regions	0.0005624772718142896	0.0006207411050636857
Fraction of Reads in promoter regions	0.26387206601482577	0.2909541854198001
Fraction of Reads in enhancer regions	0.42023376760338366	0.37593000414063676

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	215168153	57640598
Distinct Fragments	185516077	51090993
Positions with Two Read	17950770	4915230
NRF = Distinct/Total	0.862191	0.886372
PBC1 = OneRead/Distinct	0.880002	0.889522
PBC2 = OneRead/TwoRead	9.094568	9.246073

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	239704	150470
N1	280753	203082
N2	234589	154780
Np	282143	206765
N optimal	282143	206765
N conservative	239704	150470
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	1.17704752528118	1.374127733102944
Self Consistency Ratio	1.196786720604973	1.312068742731619
Reproducibility Test	pass	pass

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	299709	289749

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	303.0	262.0	521.0	349.0
50 percentile (median)	582.0	506.0	855.0	656.0
75 percentile	1028.0	966.0	1281.0	1120.0
Max size	4503.0	3555.0	4126.0	4126.0
Mean	727.200017350163	671.8459459739291	941.6516044785143	791.3609162729538

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	19.189351933972066	20.38705397658804

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.11044221683795566	0.11479228523280666
Synthetic AUC	0.4980244380963665	0.4963040114308088
X-intercept	0.09676903390914907	0.14057301663467692
Synthetic X-intercept	0.0	0.0
Elbow Point	0.8645413467690339	0.846521113243762
Synthetic Elbow Point	0.5005474626411816	0.5059644289629819
Synthetic JS Distance	0.6132654210924586	0.5879271674713874

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.5750112646536091	0.5111033054409699	0.5638510057713879	0.4957058860243961	0.5690652702070876	0.49711955020438364	0.5649038139593828	0.5538455057110997	0.5591735849053686

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.5298256567852088	0.5670392312768315	0.48985230941405167	0.5578980167250391

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.45358008237945885	0.5233459314251137	0.4417520603261042	0.5173627094282016

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates